Measurement of platelet aggregation in ovine blood using a new impedance aggregometer

Background: Whole blood platelet aggregometry (impedance) is an important method to investigate platelet function disorders. Examination of hemostatic function in sheep is important with respect to their role as an animal model of human disease. Objective: The aim of this study was to evaluate and optimize selected methodological aspects (anticoagulant, agonist concentration) of impedance aggregometry in ovine blood using the new Multiplate 5.0 analyzer. Methods: Blood samples were collected in hirudin anticoagulant from 40 clinically healthy sheep. Samples from selected sheep were collected in citrate, with or without the addition of calcium chloride. The agonists adenosine diphosphate (ADP), collagen, ristocetin, arachidonic acid, and thrombin receptor‐activating peptide (TRAP) were added in several concentrations to induce aggregation. Results: Based on maximum aggregation values and internal precision, no significant difference was found between ADP concentrations of 3–10 μmol/L and collagen concentrations of 3–5 μg/mL (P>.05). The lowest interindividual variation of approximately 3–4‐fold was seen with 4 and 5 μmol/L ADP and 4 and 5 μg/mL collagen. Ristocetin, arachidonic acid, and TRAP did not induce significant aggregation at any concentration. Aggregation results were significantly lower when measured in citrate‐ vs hirudin‐anticoagulated blood, regardless of the presence of calcium chloride. Conclusions: Our results indicate that the multiplate impedance aggregometer is suitable for the measurement of platelet aggregation in sheep using optimal agonist concentrations of 4–5 μmol/L ADP and 4–5 μg/mL collagen. Hirudin‐anticoagulated blood is the preferred sample material.     

小儿肺炎合并SIRS时血糖、CRP及PLT的改变及意义

目的 :探讨肺炎患儿 C反应蛋白 (CRP)、血小板计数 (PL T)和血糖在全身炎症反应综合征 (SIRS)中的改变及其意义。方法 :动态测定 58例肺炎合并 SIRS患儿 (SIRS组 )的 CRP、PL T和血糖 ,同时与非 SIRS的肺炎患儿 (非 SIRS组 )和正常儿童 (对照组 )作对照。结果 :非 SIRS组的血糖、PLT与对照组比较差异无显著性 (P>0 .0 5) ,而 CRP水平显著高于对照组 (P<0 .0 5) ;SIRS组血糖、CRP均显著高于非 SIRS组 (P<0 .0 1 ) ,PL T显著低于非 SIRS组 (P<0 .0 1 )。符合诊断标准 3项以上的患儿 48h血糖和第 5天 CRP持续不降 ,72 h PL T明显下降 ,多器官功能不全综合征 (MODS)发生率明显增高。结论 :联合检测肺炎患儿的血糖、CRP和 PL T计数有助于准确评估病情的危重度 ,血糖、CRP持续性升高、PLT持续下降 ,预示有发生 MODS的可能。     

海风藤酮对兔胚胎发育及PAF效应的影响

海风藤团为血小板激活因子PAF的特异性受体桔抗剂。用含20μg·mL-1海风藤酮的培养液培养兔2细胞胚胎144h,发育到等于或小于16细胞的胚胎比例为66．9％,对照组为13．5％（P＜0．025）。发育到襄胚和孵化囊胚的比例,对照组为39．9％,而试验组发育到囊胚的比例为2．7％（P＜0．001）,没有胚胎发育到孵化囊胚阶段。试验表明海风藤团对胚胎的体外发育有阻滞作用。试验还表明,兔胚胎可以释放PAF,海风藤国能明显抑制免胚胎培养液所引起的去牌鼠血小板数量下降的PAF效应。     

Preliminary investigations on the effects of a Strongylus vulgaris larval extract,mononuclear factors and platelet factors on equine smooth muscle cells in vitro

Factors involved in the proliferation of equine vascular smooth muscle cells were studied in vitro. The most prominent proliferative responses in cultured vascular smooth muscle cells were induced by Strongylus vulgaris larval antigen extract (LAE) and platelet-derived factors. Less significant proliferative responses were obtained with conditioned media from S. vulgaris LAE stimulated and from unstimulated equine mononuclear leukocytes. Additionally, vascular smooth muscle cells exposed to S. vulgaris LAE developed numerous perinuclear vacuoles and were more spindle-shaped than control or smooth muscle cells exposed to other factors. Equine mononuclear leukocytes exposed to LAE developed prominent morphological changes, including enlargement, clumping and increased numbers of mitotic figures.     

Detection of antiplatelet antibody: comparison of platelet immunofluorescence,agglutination, and immunoinjury tests using rabbit antiequine platelet serum

Immunofluorescence, tube agglutination, and platelet factor-3 immunoinjury tests for detecting antiplatelet antibody were compared using a heterologous system of equine platelets and rabbit antiequine platelet serum. Platelet immunofluorescence tests were performed using paratormaldehyde-fixed platelets in suspension as well as in air-dried smears on glass slides (solid phase). Bright homogeneous, membranous, specific fluorescence was seen in both assays with anti-immunoglobulin G (IgG) and protein G fluorescein isothiocynate conjugates (FITC-conjugates). Protein A conjugate gave nonspecific fluorescence irrespective of normal or antiserum treatment. Anti-IgG and protein G conjugates in suspension immunofluorescence tests with the same antiserum yielded antibody titers of 1:1024 and 1:128, respectively. Similarly, respective titers of 1:512 and 1:64 were obtained with solid phase immunoassay. Platelet suspension assay was slightly better than the solid phase assay. These observations indicated that anti-IgG was more sensitive than protein G in detecting antiplatelet antibody by fluorescence microscopy, while protein A was ineffective because of its nonspecificity. Chloroquine treatment of platelets failed to reduce the nonspecific fluorescence. Platelet agglutination and platelet factor-3 tests were relatively less sensitive to detect equine antiplatelet antibody.     

Effect of hemolysis on canine kaolin‐activated thromboelastography values and ADVIA 2120 platelet activation indices

Background: The impact of hemolysis on thromboelastography (TEG) and platelet activation indices has not been evaluated. Objective: The aim of this study was to investigate the influence of hemolysis induced mechanically (HM) and hemolysis induced by freezing (HF) on TEG, platelet counts (PLT), and platelet activation indicators. Methods: Blood from 17 dogs was divided into the following samples: controls, HM, and HF. HM was induced by 20 repetitions of expulsion of blood through a 23 g needle. Freezing was at −80°C, followed by warming to 37° and dilution with equal parts room temperature blood at 22°C. TEG variables that were examined included reaction time (R), coagulation time (K), angle (α), maximum amplitude (MA), and clot rigidity (G). Platelet indices were measured with the ADVIA 2120 hematology analyzer. Results: Hematocrit (HCT) (mean±SD) for controls, HM, and HF were 0.41±0.02, 0.39±0.03, and 0.25±0.02 L/L, respectively, consistent with decreases in HCT of 4.8% (HM) and 39.0% (HF). HM resulted in decreased R (2.5±0.9 minutes compared with 5.2±1.9 minutes for controls; P<0.001), and HF resulted in increased K (15.2±8.6 minutes compared with 5.3±4.0 minutes in controls; P<0.01) and decreased α (20±11° compared with 46±17° in controls; P<0.001). MA was decreased more in HF samples (26±2 mm) than in HM (38±8 mm) or control samples (49±9 mm; P<0.0001). The same applied to G values. PLT decreased after HM but not after HF. Hemolysis of both types resulted in decreased mean platelet component (MPC) concentration: control, 19.3±2.0, HM 15.5±3.4, and HF 14.3±0.7 g/dL (P<0.0001). Conclusion: In hemolyzed samples decreased MPC and R suggested activated primary and secondary hemostasis, respectively, but decreased MA and G indicated reduced clot firmness, possibly due to hyporeactive platelets. TEG and platelet activation indices should be interpreted cautiously after hemolysis.     

汇集滤白细胞血小板的研制及临床应用

目的 评价汇集滤白细胞血小板的质量和临床效果。方法用白膜层法制备手工浓缩血小板，再以60～70mL／min的速度对汇集血小板进行白细胞过滤。30袋汇集滤白细胞血小板过滤前后分别进行血小板含量、白细胞残留量检测和细菌培养，并观察87例出血患者（血液病16例、非血液病71例）输注后24h疗效。结果30袋汇集滤白细胞血小板过滤前后血小板含量差异无统计学意义（P〉0．05），白细胞残留量差异有统计学意义（P〈0．01），细菌培养均为阴性。非血液病出血患者输注汇集滤自细胞血小板的有效率是85．9％，而血液病患者输注有效率是75．0％。结论汇集滤白细胞血小板质量指标均达到单采血小板国家质量标准，临床应用效果良好。